The goal of this project is to characterize the gating currents and gating mechanisms of the hyperpolarization-activated cyclic nucleotide-gated cation channel (HCN) subfamily of voltage-gated ion channels. HCN channels are structurally similar to voltage-gated K+ (Kv) channels, but open in response to nembrane hyperpolarization instead of depolarization. Voltage-gated ion channel opening can be described by three processes: voltage sensing, allosteric coupling and channel activation or pore opening. This project aims to understand the molecular relationship between voltage sensing and the allosteric coupling that causes HCN channel activation. In Kv channels, voltage sensing arises from the voltage dependent movement of the positively charged, transmembrane domain S4. Movement of S4 results in pore opening or closing and can be measured by the gating current it produces. This project will determine how gating currents are associated with he time- and voltage-dependent gating of HCN channels, how the S4 domain contributes to these gating currents, and how the S4-S5 linker affects these gating currents and couples them to pore opening. This proposal will provide the first direct measure of the role that S4 plays in HCN channel activation. It will elucidate the magnitude, direction, voltage dependence and kinetics of HCN gating current, determine which residues in S4 contribute to gating current and how the S4-S5 linker modulates S4 movement and couples it to HCN channel opening.